Many of the world's most important infectious diseases are transmitted to humans by insect vectors. Attempts to eliminate the insect vectors or to immunize and protect the human host have thus far been unsuccessful. Alteration of insect vector competence is an alternative strategy to interrupt the cycle of disease transmission. Preliminary data from the Principal Investigator's laboratory document the unique capability of retroviral vectors containing the envelope glycoprotein of vesicular stomatitis virus (VSV-G) to infect and stably integrate into a broad range of non-mammalian cells including fish, frog, newt, and insect cells. These pantropic, replication-incompetent vectors have been used to create transgenic fish with stable, Mendelian segregation of the provirus in the F2 generation. The collaborating investigators have demonstrated that microinjection of mosquito eggs with concentrated viral stocks results in a high percentage of adult transformants containing the provirus. Using a Sindbis vector expression system, the collaborating investigators have shown protection of mosquito cells expressing the nucleocapsid gene of flaviviruses from superinfection with the homologous viral strain. These data suggest that pantropic retroviral vectors can be used to genetically modify insects to prevent transmission of important human pathogens. The proposed project will bring together three different investigators with established expertise in mosquito vector biology, virology, and retroviral vector design with the following goals: 1) to develop VSV-G pseudotyped retroviral vectors that mediate stable gene expression in mosquito cell lines and in transgenic progeny of germline transformants and 2) to create transgenic mosquitoes with a pantropic retroviral vector expressing the nucleocapsid gene of different flaviviruses, to confer cellular resistance to superinfection following challenge with wild type virus. The current proposal is a unique opportunity to develop a transformation system for the mosquito vector community that will permit the testing of strategies to alter mosquito vector competence to prevent vector-borne disease transmission.